PROJECT SUMMARY By feeding on human blood, mosquitoes annually infect hundreds of millions of people with disease. Understanding how they identify and home in on their hosts represents an unparalleled opportunity to decipher the behavioral strategies of one of the most successful predators in evolutionary history, and potentially improve public health and quality of life worldwide. Aedes aegypti offers a unique, genetically accessible system to study how human odor is sensed by mosquitoes. Mutants that ablate olfactory receptor function of the three olfactory receptor families in mosquitoes are available. These mutants have demonstrated that olfaction is critical for mosquitoes to sense their human hosts and avoid repellents. We have developed a technique that allows us figure out which genes ?smell? an odor by assessing how the olfactory receptor gene expression is altered after a mosquito is exposed to that odor. By exposing mosquitoes to human odor and single human odor components, we will find the genes that enable mosquitoes to find their human hosts. This proposal uses a new technique developed in mammals and Drosophila, Deorphanization of Receptors based on Expression Alteration of mRNA levels (DREAM). Our unpublished data demonstrates the efficacy of this technique in mosquitoes. This will allow us to comprehensively determine which Aedes aegypti mosquito olfactory receptors are activated by human odor in vivo by characterizing the reduction of mRNA levels of these receptors using RNA-seq and quantitative RT-PCR. The genes identified with our approach are likely to be odor-gated ion channels such as Odorant Receptors (ORs) or Ionotropic Receptors (IRs). The identified ORs and IRs can then be used to screen for chemicals that can activate them with higher affinity than their natural odor-ligands. Alternatively, chemicals that block their activation in the presence of their odor-ligands can become lead compounds to develop new repellents. As RNA-seq does not use gene-specific primers to generate sequence data, this approach may also reveal genes not previously thought to be involved in the sensation of human odor by mosquitoes. In addition, the approaches developed by this proposal can be applied to understanding host detection mechanisms in other vectors of human disease such as Glossina morsitans, Ixodes scapularis, and other mosquito species such Aedes albopictus, Anopheles gambiae and Culex quinquefasciatus. The conservation of DREAM across both vertebrates and invertebrates makes it potentially a powerful tool for understanding chemosensation in many organisms. Thus, our approach will provide insight into vector biology in three ways: the molecular mechanism of mosquito human host detection, technical information that can be applied to the understanding of chemosensation in other vectors, and a list of molecular targets for mosquito attractant and repellent design.